The need to isolate particular molecules, e.g., proteins, has long been known. Moreover, various protocols exist by which molecules may be isolated. For example, in gel electrophoresis proteins are placed in the middle of a buffered gel (e.g., polyacrylamide gel) between oppositely charged electrodes. When the electrodes are charged, each of the protein molecules travels toward one of the electrodes, according to their net charge at the pH of the buffered polyacrylamide gel. The speed at which the protein molecules move through the gel toward the electrodes is largely dependent on the size of the molecule, i.e., smaller molecules move faster through the gel matrix. As a result in the differences in speed, types of protein molecules can be separated and then isolated.
A variant to gel electrophoresis is isoelectric focusing, which exploits the fact that the net charge of a protein depends on the environmental pH. Most generally, at acidic pH, proteins are globally positively charged while in alkaline pH they are negatively charged. The pH at which the protein has no net charge is called the isoelectric point (“pI”). Isoelectric focusing is an electrophoresis technique in which proteins move under an electric field through a pH gradient. All proteins migrate towards the cathode or the anode until they encounter a pH identical to their isoelectric point. At this point the protein loses its charge and stops moving. Proteins of different isoelectric points stop at different levels and are thus separated. Accordingly, similarly sized molecules, which may move at similar speeds, can be separated after coming to rest at different pH points, as a result of having different pI values. In addition, there are situations in which migration by the size in a given buffered gel and migration by the isoelectric point are crossed for an enhanced separation of protein species from very complex mixtures; the technique used in this situation is called bidimensional electrophoresis. Unfortunately, migration of proteins within an electrophoresis gel network according to these techniques is a very slow process and is generally unacceptable for preparative purposes.
In response, various additional protocols have been developed which have attempted to increase the rate of separation, while preserving the accuracy by which it is performed. There are many types of devices comprising two or more subcompartments that are separated from each other by septa, e.g., monofilament screens, membranes, gels, filters, fritted discs, and the like (collectively, “membranes”). Generally, these devices are assembled from a plurality of essentially parallel frames or spacers, separated from each other by one or more membranes.
Multi-compartment electrolizers with isoelectric membranes were introduced for processing large volumes and amounts of proteins to homogeneity. For example, see P. G. Righetti, et al., “Preparative Protein Purification in a Multi-Compartment Electrolyser with Immobiline Membranes,” 475 J. CHROMATOGRAPHY 293-309 (1989); P. G. Righetti, et al., “Preparative Purification of Human Monoclonal Antibody Isoforms in a Multi-Compartment Electrolyser with Immobiline Membranes,” 500 J. CHROMATOGRAPHY 681-696 (1990); P. G. Righetti, et al., “Preparative Electrophoresis with and without Immobilized pH Gradients,” 5 ADVANCES IN ELECTROPHORESIS 159-200 (1992). Based on isoelectric focus, this purification concept progresses under recycling conditions. The protein macro-ions are kept in a reservoir and are continuously passed through an electric field across a multicompartment electrolyzer equipped with zwitterionic membranes.
In this system the protein is always kept in a liquid vein, also called a “channel.” Consequently, the protein is not lost by adsorption onto surfaces, as typically occurs in chromatographic procedures. Rather, the protein is trapped in a chamber that is delimited by two membranes which have pI values encompassing the pI value of the protein to be separated. Thus, by a continuous titration process, all other impurities, either non-isoelectric or having different pI values, are forced to leave the chamber. In the end, the isoelectric/isoionic protein of interest will ultimately be present, as the sole species, in the chamber. It should be recognized, however, that the isoelectric and isoionic points of a protein can differ to some extent in the presence of counterions.
U.S. Pat. No. 4,971,670 describes this process. Isoelectric membranes also are addressed in U.S. Pat. No. 4,243,507. U.S. Pat. No. 5,834,272 describes an immobilization of enzymes that keeps them in solution and, hence, under conditions of homogeneous catalysis.
In U.S. Pat. No. 4,362,612, adjoining compartments are functionally designed to adjust to different pH values electrophoretically, thereby separating dissolved proteins according to their isoelectric points. Similar multiple subcompartments devices are described in U.S. Pat. Nos. 4,971,670, 5,173,164, 4,963,236, and 5,087,338. Each of these patents discloses a device comprised of a series of parallel spacers, separated from each other by membranes, that provides an essentially parallel array of subcompartments. Similarly, Amersham Pharmacia created an IsoPrime filter using a plurality of pI-selective membranes arranged in series. In this device the membranes are arranged in ascending or descending pI-selectivity. As a solution passes through the membranes, molecules having pI values between two consecutive membranes are trapped between the membranes. However, this process takes on the order of hours to complete. Invitrogen, Inc. invented a device, the ZOOM IEF Fractioner, which is substantially similar to the IsoPrime device, but which enables the membranes to be individually replaced. However, like the IsoPrime, the ZOOM IEF Fractioner process takes on the order of hours to complete.
What is needed, therefore, is an apparatus and a methodology that address at least one if not more of the deficiencies that afflict conventional practice, as previously described. More particularly, the need exists for an approach for separating molecules, such as proteins, quickly and accurately accordingly to pI values.